Synthesis and evaluation of in vitro and in vivo anti-Toxoplasma gondii activity of tetraoxane-substituted ursolic acid derivatives

Abstract A series of derivatives of ursolic acid (UA) were synthesised, the anti-Toxoplasma gondii activity was tested, and the selectivity index (SI) of these compounds was calculated to determine the derivative with the best anti-Toxoplasma gondii activity. Compound A7 showed the best activity against the Toxoplasma gondii (IC50 in T. gondii infected GES-1 cells: 9.1 ± 7.2 μM), better than the lead compound UA and the positive control drug Spiramycin. Compound A7 was selected for further in vivo research: A7 was tested for its effect on the inhibition rate of tachyzoites in mice and its biochemical parameters, such as alanine aminotransferase, aspartate aminotransferase, glutathione, and malondialdehyde were determined. Compound A7 was evaluated for its anti-Toxoplasma activity and partial damage to the liver. Therefore, the results show that compound A7 could be a potential lead compound for developing a novel anti-Toxoplasma gondii molecule. Graphical Abstract


Introduction
Toxoplasmosis is a globally transmitted disease that is diagnosed in approximately one-third of the population worldwide (Luder et al. 2001).Toxoplasma gondii can infect all warm-blooded animals and even some cold-blooded animals and parasitise all nucleated cells (Djurkovic-Djakovic et al. 2019).Toxoplasmosis has a wide range of transmission routes, and the host can be infected through diet, vertical transmission, organ transplantation, and blood transfusion (Shapiro et al. 2019).In individuals with strong immunity, toxoplasmosis usually shows no symptoms or is a mild, self-limiting infection.However, toxoplasmosis can be life-threatening in fetuses and newborns with underdeveloped immune systems and in those with HIV or other diseases that compromise cellular immunity (Osunkalu et al. 2011).There is no effective vaccine for toxoplasmosis, and chemotherapy is mostly used in clinical treatment.Classic treatment for toxoplasmosis usually protect organs from damage by inhibiting the replication of parasites but cannot altogether remove them from the host body.Clinically, almost all treatment options are inseparable from sulfa drugs, but these drugs often cause allergies and are accompanied by blood toxicity.
Most natural products have good pharmacological activity and low toxicity, and a large number of studies have also reported that natural products have anti-T.gondii activity (Deng et al. 2022 ).Ursolic acid (UA) is a natural product of five ring triterpenoids, as shown in Figure 1.Ursolic acid has anti-inflammatory (Wang et al. 2021;Zhang et al. 2022) and antibacterial (Wu et al. 2019) activity anti-T.gondii activities (Choi and Lee 2018).
Tetraoxane has peroxy bonds and has been found to have certain anti-T.gondii effects (Xin et al. 2016).Previous studies found that N-251, a compound containing oxygen bridges, has an anti-Toxoplasma gondii effect (Xin et al. 2016).In vivo anti-T.gondii studies with tetraoxane compounds have found that tetraoxanes have no obvious toxicity and can also significantly prolong the survival period of mice infected with T. gondii, and the therapeutic effect is even higher than that of artemisinin (Opsenica et al. 2015).Previous studies have shown that modifying the carboxyl group of UA greatly improves its anti-Toxoplasma gondii activity (Luan et al. 2019).Therefore, we used UA as the lead compound, introduced tetraoxane as a fragment into its carboxyl group, and synthesised a series of compounds to obtain target compounds with strong anti-T.gondii activity and acid derivatives.

Chemistry
A series of 18 UA derivatives were synthesised.The synthetic route for the target compound is shown in Scheme 1.The final products obtained were all white powders, and according to experimental calculations, the yield of the compound was 25-42%.

Evaluation of biological activities
2.2.1.Evaluation of in vitro anti-T.gondii activity (MTT assay) All compounds were tested for toxicity and anti-T.gondii activity and the SI values were calculated (Table S1).According to the data, almost all compounds showed better anti-T.gondii activity than UA and spiramycin.Among them, compound A7 had the best activity, and its IC 50 value against T. gondii was 9.1 ± 7.2 lM and SI value was 6.4.
From the IC 50 of compounds A16-A18, it was found that the linker length between UA and the tetraoxane fragment has a certain effect on the activity of the compound to inhibit T. gondii.Among them, the two-carbon linker compound A16 showed the best activity.As the length of the linker increased, the activity of the compound against T. gondii decreased.
The different substituents on the benzene ring of compounds A4-A15 also had certain effects on the compounds.Surprisingly, when the substituent on the benzene ring was chlorine, it showed better anti-T.gondii activity than the other halogen atoms.The ortho position was the most active among the compounds with the same substituents, followed by the para and meta positions.

Number of tachyzoites in vivo
The number of tachyzoites, shown in Table S2, in the abdominal cavity of mice was evaluated to determine whether the compound had an inhibitory effect on it.
The results showed UA and A7 administered intragastrically to infected mice.The inhibition rates of UA and A7 on T. gondii in the abdominal cavity were 21.6% and 54.2%, respectively, which were significantly different from those in the Toxo group ( Ã p < 0.05, ÃÃ p < 0.01), and the inhibitory effect of A7 was significantly stronger than that of Spi ( && p < 0.01).These results indicate that compound A7 has a significant inhibitory effect on the number of tachyzoites in the abdominal cavity of mice.

Mouse body weight index
Mice infected with T. gondii had a significant loss of appetite which leads to the weight loss of the mice.The data (Figure S2) showed that the bodyweight of mice infected with T. gondii was significantly reduced ( ### p < 0.001).UA, Spi, and A7 had a relieving effect on weight loss caused by T. gondii infection.Compound A7 significantly increased the weight of the infected mice ( ÃÃ p < 0.01), and there was no significant difference from that of the normal group, indicating that compound A7 can significantly improve the problem of decreased appetite caused by T. gondii infection 2.2.4.Liver and spleen indices T. gondii infection causes a series of pathological changes, including hepatomegaly (Hassan et al. 1996).T. gondii reproduces in liver cells, causing hepatocyte necrosis accompanied by splenomegaly (Bourguin et al. 1993;Xu et al. 2019).
Hepatomegaly was observed in mice infected with (Figure S3) T. gondii compared with those in the normal group.Compound A7 failed to cause significant changes in the liver index of the mice infected with T. gondii.However, compared with the normal group, the infection with T. gondii significantly increased the spleen index of the mice infected with T. gondii, indicating that the infection causes obvious splenomegaly.Compound A7 significantly inhibited splenomegaly in the infected mice ( ÃÃÃ p < 0.001), exhibited a stronger inhibitory effect on splenomegaly than lead UA ( ÃÃ p < 0.01), and was slightly better than that of Spi ( ÃÃÃ p < 0.001).

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels
To further study the toxicity of the tested drugs on experimental mice, we measured alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the serum of the mice.When liver cells were damaged, liver function and metabolism were reduced, and incomplete metabolites were produced, increasing the liver index.The liver index reflects the degree of liver damage (Michalopoulos 2007).ALT and AST are non-specific enzymes in the liver.When liver cells are damaged, the levels of ALT and AST increase significantly (Zhang et al. 2016).Liver damage caused by T. gondii can lead to an increase in ALT and AST in the body (Mordue et al. 2001).
After infection with T. gondii, the levels of ALT and AST in the blood of the host increased significantly (Figure S4).The results showed that A7 significantly reduced the levels of ALT and AST in the blood of infected mice ( ÃÃ p < 0.01, ÃÃÃ p < 0.001), and the inhibitory effect of compound A7 on ALT was better than that of UA and Spi.
Moreover, AST levels in the blood of infected mice in the A7 group were not significantly different from those in the normal group.The inhibitory effect of compound A7 on AST was better than that of the lead compound and the positive control drug.

Glutathione (GSH) and malondialdehyde (MDA) levels
Toxoplasma gondii infection can lead to oxidative stress and immunosuppression (Elsheikha et al. 2009;Xu et al. 2019).Oxidative stress plays an important role in liver damage caused by T. gondii, and is accompanied by an increase in malondialdehyde (MDA) levels.MDA reflects the degree of lipid peroxidation, which is related to liver lipid peroxide damage.Excessive reactive oxygen species (ROS) can cause cell and tissue damage (Xu et al. 2019;Smith and Shanley 2013).During T. gondii infection, ROS production increases.Antioxidant enzymes can inhibit oxidative damage and produce ROS (Ebisch et al. 2007;Xu et al. 2019).By eliminating superoxide anion free radicals, proteins such as glutathione (GSH) can protect cells from oxidative damage.GSH levels in mice exposed to T. gondii were significantly reduced (Ebisch et al. 2007).
Acute infection with T. gondii decreases the antioxidant capacity of the host, decreases GSH content in the liver, and increases MDA content.The results showed that after A7 was administered intragastrically to infected mice, the level of GSH in the liver of infected mice increased significantly ( ÃÃ p < 0.01), compared with the Toxo group, and compound A7 showed a better improvement in the GSH levels than UA ( Ã p < 0.05), and the effect was similar to that of Spi.At the same time, compound A7 significantly reduced the level of MDA ( Ã p < 0.05), and its effect on MDA was significantly better than that of UA and Spi (shown in Figure S5).

Chemistry procedure for compounds a1-a18
Substitute carbonyl compound (1 mmol) and 30% H 2 O 2 (10 mmol) were dissolved in acetonitrile (10 mL) and dichloromethane (DCM) (30 mL), then catalytic amount of concentrated hydrochloric acid was added, and the solution was stirred at room temperature for 4-8 h.The reaction was monitored by TLC and the reaction mixture was neutralised with NaHCO 3 and extracted with dichloromethane and ethyl acetate.Then, it was dried over anhydrous Na 2 SO 4 , and evaporated in vacuo.

Chemistry procedure for compound UA-1
To a solution of ursolic acid (1eq) and 1,2-dibromoethane (3eq) or 1,3-dibromopropane(3eq) or 1,4-dibromobutane (3eq) in acetonitrile (25 mL), K 2 CO 3 (1eq) was added and then the mixture was heated to reflux for 8 h and monitored by TLC.After completion of the reaction, the solvent was extracted three times with DCM and the combined organic layer was washed with brine, dried over MgSO 4 , and evaporated to dryness.The crude product was purified through column chromatography (Petroleum ether: ethyl acetate ¼ 30:1).

Chemistry procedure for compound UA-2
To a solution of UA-1 (1eq) and p-hydroxybenzaldehyde (3eq) in acetonitrile (20 mL), K 2 CO 3 (1eq) was added.The mixture was refluxed for 24 h.When TLC monitoring shows that the reaction is complete, extracted with DCM and the organic phase was washed with saturated sodium hydroxide solution, and brine and dried over anhydrous Mg 2 SO 4 .The evaporation of the solvents gave the crude products, which were purified by silica gel column chromatography (Petroleum ether: ethyl acetate ¼ 30:1) to afford compounds UA-2.

Chemistry procedure for compounds A1-A18
To a solution of crude product of a1-a18 (1eq) and the UA-2 (1eq) in DCM (20 mL) was added concentrated sulfuric acid diluted with acetonitrile slowly at 0 C (MeCN: H 2 SO 4 ¼10:1, V/V) .The mixture was stirred for 0.5 h in an ice bath.When TLC monitoring shows that the reaction is complete, the reaction mixture was neutralised with NaHCO 3 and extracted with dichloromethane and the organic phase was washed with saturated brine and dried over anhydrous Mg 2 SO 4 .The evaporation of the solvents gave the crude products, which were purified by silica gel column chromatography (Petroleum ether: ethyl acetate ¼ 20:1) to afford compounds A1-A18.

Evaluation of anti-T. gondii activity in vitro (MTT assay)
The reported methylthiazolyltetrazole (MTT) method was used to study the cytotoxicity of compounds on host cells: Seed the human gastric epithelial cells (Ges-1) in a 96well plate at an appropriate density to ensure that the cells grow exponentially (3000 cells/well).Then let them adhere for 24 h at 37 C. Infect Ges-1 cells with Toxoplasma gondii (15,000 tachyzoites/well), and then culture the Ges-1 cells infected with Toxoplasma gondii for 24 h.Dissolve the 10 mM compound in DMSO and store it, and test its serial dilution (1-500 lM), and spiramycin was used as positive controls.After incubating the plate for 24 h, add 10 lL of MTT solution to each well.Then incubate for another 4 h.Read the optical density (OD) from the microplate reader at a wavelength of 540 nm. and the median inhibitory concentration of compounds on Ges-1 cells infected with Toxoplasma gondii in vitro.Then calculate the selectivity index (SI), The cytotoxicity of the compound was simultaneously measured under the same experimental conditions, and the operation method was roughly the same, the only difference was that Ges-1 cells were not infected by Toxoplasma gondii.After the final determination of the OD value, the IC 50 of each group of cells were calculated, and then the selectivity index (SI) of the compound was obtained.

Evaluation of anti-T. gondii activity in vivo
Healthy female KM mice (g) were randomly divided into 5 groups, 6 animals per group, each group namely the normal group, the Toxo group, the Spi group, the compound A7 group and the UA group.Except that the normal group was not injected with T. gondii, the other groups were injected with 2 Â 10 3 T. gondii strains intraperitoneally, and were administered separately after infection for 4 h.The normal group and the control group were intragastrically administered 0.2 mL of normal saline.The spi group, the UA group, and the compound A7 group were given intragastrically at a dose of 100 mg/kg, once a day for four consecutive days.After weighing the weight of each group of mice, blood was collected from the heart, mice killed by cervical dislocation.Then, the abdominal cavity of the mice was washed with an appropriate amount of sterile normal saline, the abdominal cavity fluid was collected and the parasites were centrifuged ten minutes.The number of T. gondii in the abdominal cavity of each group of mice was counted by a hemocytometer.After each group of mice was sacrificed by neck dissection, the mice's liver and kidney were dissected, and the mouse liver and kidney were separated.Weighed and recorded the weight of each mouse's liver and spleen, and then calculated Liver and spleen index.Using appropriate amount of serum and measure ALT (Guo et al. 2019) and AST (Zhang et al. 2018), using liver tissue to calculate GSH (Zhang et al. 2021) and MDA (Shang et al. 2019(Shang et al. , 2021)).This study is approved by animal ethical committee to conduct the experiments as per standard guidelines.

Conclusions
In this experiment, 18 ursolic acid derivatives were synthesized.Compound A7 had the best anti-T.gondii activity with an IC 50 value of 9.1 ± 7.2 lM, which is 37 times higher than that of UA (336.2 ± 12.0 mM) and 24 times higher than that of spiramycin (216.5 ± 17.1 mM).According to the in vivo experimental data analysis, compound A7 could significantly inhibit the number of tachyzoites in mice and significantly improve the symptoms of loss of appetite in mice infected with T. gondii.In addition, compound A7 had a strong protective effect on the liver.It also alleviated splenomegaly in infected mice with minimal side effects.In summary, compound A7 is a candidate anti-T.gondii agent with great application potential.